Analysis of Aminoglycoside Antibiotics: A Challenge in Food Control

Aminoglycosides are a widely used group of antibiotics in veterinary medicine. However, misuse and abuse of these drugs can lead to residues in the edible tissues of animals. Due to the toxicity of aminoglycosides and the exposure of consumers to the emergence of drug resistance, new methods are being sought to determine aminoglycosides in food. The method presented in this manuscript describes the determination of twelve aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, neomycin, gentamicin, hygromycin, paromomycin, kanamycin, tobramycin, amikacin, apramycin, and sisomycin) in thirteen matrices (muscle, kidney, liver, fat, sausages, shrimps, fish honey, milk, eggs, whey powder, sour cream, and curd). Aminoglycosides were isolated from samples with extraction buffer (10 mM NH4OOCH3, 0.4 mM Na2EDTA, 1% NaCl, 2% TCA). For the clean-up purpose, HLB cartridges were used. Analysis was performed using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with a Poroshell analytical column and a mobile phase of acetonitrile and heptafluorobutyric acid. The method was validated according to Commission Regulation (EU) 2021/808 requirements. Good performance characteristics were obtained for recovery, linearity, precision, specificity, and decision limits (CCα). This simple and high-sensitivity method can determine multi-aminoglycosides in various food samples for confirmatory analysis.

Aminoglycoside antibiotics are among the most common antibiotics in animal husbandry to treat severe bacterial infections. They are applicable against infections caused by Gram-negative and also Gram-positive organisms. Improper use of drugs, failure to observe the withdrawal period, or slaughtering animals during treatment can result in drug residues in the tissues. Due to extended withdrawal times and high residue levels, using these drugs as veterinary medicines carries the risk of developing resistance among bacteria, which may reduce the effectiveness of these drugs as human medicines [3]. Nearly all of the aminoglycosides are widely used in human and veterinary medicine. The residues of This study aimed to develop one all-purpose method for food matrices. It is the first time an analytical method has been described for determining twelve aminoglycoside antibiotics in a wide range of matrices (honey, eggs, tissues, meat, and milk products) not presented in other publications. In addition, a significant challenge was the determination of one method of compounds without MRLs (low decision limit (CCα) required) as well as compounds with high MRL values (wide range of method linearity required).

Optimization of LC-MS/MS Conditions
Aminoglycosides are polar compounds due to several amino groups contributing to their weak basic nature [37]. Confirming the presence of aminoglycosides at trace residue concentrations using ultra-high-performance liquid chromatography coupled with the tandem mass spectrometry (UHPLC-MS/MS) method has been a challenge for a long time. A notable problem was the separation of aminoglycosides since some have similar structural forms and mass, such as DHSTR, STR, PAR, and NEO. Detection was performed in multiple reaction monitoring (MRM) modes by directly infusing the standard analyte solution into the mass spectrometer. Two transitions were monitored for each analyte, except ribostamycin as an internal standard with one transition. Three transitions were monitored for aminoglycosides with similar retention times or the same ions for better identification and chromatographic separation. The additional transitions were performed for AMI, DHSTR, KAN, NEO, PAR, and STR for quantification to serve the most abundant precursor ion transitions. In contrast, other transitions have been used for identification. The following mass spectrometry parameters, in positive ionization mode, were designated: collision energy (CE), declustering potential (DP), dwell time, and cell exit potential (CXP) of each compound (Table 2). To achieve optimal separation of aminoglycosides and UHPLC-MS/MS quantification as well as confirmation, reversed-phase C18 chromatographic columns were tested-Poroshell 120 EC-C18 (2.1 × 50 mm 2 ; 2.7 µm), Agilent ZORBAX SB-C18 (50 mm × 2.1 mm, 1.8 µm), and Poroshell 120 EC-C18 (2.1 × 150 mm 2 ; 2.7 µm). The separation of aminoglycosides on the first column gave unsatisfactory results; non-symmetrical peaks were observed, particularly for GEN, SPC, and PAR. Moreover, low-intensity peaks on the ZORBAX SB-C18 column were obtained, especially for DHSTR, KAN, GEN, APR, and TOB.
The most efficient way to obtain suitable retention of these polar compounds is to use ion-pairing (IP) agents. Błądek et al. [38] compared various IP reagents, namely, heptafluorobutyric acid (HFBA), trifluoroacetic acid (TFA), and pentafluoropropionic acid (PFPA). During the optimization of chromatographic separation, different IP agents as a mobile phase (TFA, HFBA) with various combinations of gradient programs were tested. In this study, in our method, HFBA in low concentration gave the highest ionization, the best shapes of peaks, good recoveries, and a short elution time. In many multi-component works, HFBA is used as an ion-pairing agent [10,39,40]. The following parameters were tested during chromatographic optimization: the chromatographic column's temperature and the mobile phase's flow. Next, temperature was checked: 30 • C, 35 • C, and 40 • C. Different values of flow rate were optimized: 0.25, 0.30, 0.35, and 0.4 mL/min. Finally, the most satisfactory results to analyze AMI, APR, DHSTR, GEN, HYG, KAN, NEO, PAR, SIS, SPC, STR, and TOB were achieved using a longer Poroshell 120 EC-C18 (2.1 × 150 mm; 2.7 µm) column at a temperature of 35 • C with acetonitrile and 0.025% HFBA mobile phase. The flow rate was set as 0.3 mL/min. The chromatographic separation of 12 standards of aminoglycosides is presented in Figure 1.
The most efficient way to obtain suitable retention of these polar compounds is to u ion-pairing (IP) agents. Błądek et al. [38] compared various IP reagents, namely, he tafluorobutyric acid (HFBA), trifluoroacetic acid (TFA), and pentafluoropropionic ac (PFPA). During the optimization of chromatographic separation, different IP agents a mobile phase (TFA, HFBA) with various combinations of gradient programs were teste In this study, in our method, HFBA in low concentration gave the highest ionization, t best shapes of peaks, good recoveries, and a short elution time. In many multi-compone works, HFBA is used as an ion-pairing agent [10,39,40]. The following parameters we tested during chromatographic optimization: the chromatographic column's temperatu and the mobile phase's flow. Next, temperature was checked: 30 °C, 35 °C, and 40 ° Different values of flow rate were optimized: 0.25, 0.30, 0.35, and 0.4 mL/min. Finally, the most satisfactory results to analyze AMI, APR, DHSTR, GEN, HY KAN, NEO, PAR, SIS, SPC, STR, and TOB were achieved using a longer Poroshell 120 E C18 (2.1 × 150 mm; 2.7 µm) column at a temperature of 35 °C with acetonitrile and 0.025 HFBA mobile phase. The flow rate was set as 0.3 mL/min. The chromatographic sepa tion of 12 standards of aminoglycosides is presented in Figure 1.

Optimization of Sample Preparation
Based on Cherlet et al. [32,41], some extraction mixtures, in various volumes and concentrations, were tested: 10 mM ammonium acetate/0.4 mM EDTA/1% NaCl/2% TCA with 0.2 µL/0.5 µL 0.3 M HFBA; 150 mM EDTA + 5%/15% TCA. The mainly used chemical reagents for sample preparation are TCA + EDTA [32,42,43], NH 4 OOCH 3 + Na 2 EDTA + NaCl + TCA, and K 2 HPO 4 in various concentrations [30]. The best results for all aminoglycoside isolations from the matrix were obtained in the presented method after using a solution consisting of 10 mM ammonium acetate/0.4 mM EDTA/1% NaCl/2% TCA. Different TCA concentrations in the extraction solution were also tested (1%, 2%, 5%, 15%). Figure 2 shows the recovery (%) of all analyzed aminoglycosides after using different extraction mixtures. ces such as liver, honey, and milk. The Strata X-AW gave significantly better re eggs, fish, and shrimps. However, the number of compounds and matrices make essary to find a single optimal solution, so after analyzing all the columns, it was to use Oasis HLB cartridges with the best results for all tested compounds in all m For better recovery of analytes, a double elution was used with the mixture o acid/isopropanol/water (10:5:85). The very high sensitivity of aminoglycosides to change the pH value in the extraction solution makes selecting sample preparation conditions a significant challenge for the analyst. Thus, different pH values were tested (5.5, 6.5, and 8.0). Figures 3 and 4 show the effect of pH values for liver and honey samples as the most sensitive matrices. The results for other matrices are presented in the Supplementary Materials ( Figure S1). Tests show that at pH = 8.0, the peak area decreased by 50-80% in most cases. These results confirm that aminoglycosides are sensitive to pH changes and that pH = 6.5 is optimal for determining most aminoglycosides.    The next step was to select the appropriate cleaning up. Generally, extraction methods published for aminoglycoside analysis mostly used SPE extraction with Oasis HLB or Strata X cartridges. Oasis HLB is used in many laboratories because of its good retention properties and highly reproducible recovery of a wide range of compounds, both polar and non-polar (due to the combination of their hydrophobic-hydrophilic retention mechanism). In this study, Strata X (100 mg, 6 mL), Strata X-CW (100 mg, 6 mL)-cation weak, Strata X-AW (100 mg, 6 mL)-anion weak, and Oasis HLB (60 mg, 3 mL) cartridges were verified (Table 3). Strata X-CW gave the worst results for most of the matrices. The results were entirely satisfactory only in muscle, liver, and eggs. Strata X, in turn, gave the best results in honey and good results in kidney, fat, sausages, milk, and whey powder. The results between Strata X-AW and Oasis HLB cartridges were comparable for some matrices such as liver, honey, and milk. The Strata X-AW gave significantly better results for eggs, fish, and shrimps. However, the number of compounds and matrices makes it necessary to find a single optimal solution, so after analyzing all the columns, it was decided to use Oasis HLB cartridges with the best results for all tested compounds in all matrices. For better recovery of analytes, a double elution was used with the mixture of formic acid/isopropanol/water (10:5:85).

Method Validation
The developed method was validated according to the Commission Implementing Regulation (EU) 2021/808 [44]. A good linearity was obtained, and the correlation coefficients (r2) were higher than 0.98 for all analytes in each matrix. The selectivity of the test showed no interference peaks in the samples analyzed. Moreover, repeatability (CVs within 3.5 to 14.2, depending on compound and matrix) and within-laboratory reproducibility (CVs within 4.3 to 13.8, depending on compound and matrix) were satisfactory. The recovery values ranged from 82% to 118% for trueness −79-117%. For ruggedness, the factors and changes examined did not affect the results, indicating that the method developed is robust to minor changes that may occur in the study. The limits of quantifications (LOQ) were from 10 µg/kg to 250 µg/kg. The coefficients of variations are under 20% for the matrix factor (MF) in every analyte and matrix. The summary of validation is shown in Table 4. The example chromatograms (MRM) of a shrimp sample, as a matrix without MRL, fortified at a validation level (VL) of 100 µg/kg with all twelve analytes are presented in Figure 5.

Aminoglycosides in Real Samples
Nearly 750 muscle, tissue, and honey samples were tested for confirmation by LC-MS/MS methods over the past ten years in our laboratory. Of these, 129 samples (about 17%) contained aminoglycosides, of which up to 61% were non-compliant. The most fre-

Aminoglycosides in Real Samples
Nearly 750 muscle, tissue, and honey samples were tested for confirmation by LC-MS/MS methods over the past ten years in our laboratory. Of these, 129 samples (about 17%) contained aminoglycosides, of which up to 61% were non-compliant. The most frequently detected aminoglycoside antibiotics were DHSTR (49 non-compliant results, 19 compliant results) in the concentration range 60-229,000 µg/kg and NEO (17 noncompliant results, 22 compliant results) in the concentration range 256-87,000 µg/kg. Aminoglycosides were mainly detected in cattle (96 results) and swine (17 results). Although it may seem that the percentage of samples with aminoglycosides is small compared to other groups (tetracyclines), the toxicity of these compounds and the difficulty of quantifying them seems to be quite a challenge for analysts.
The method presented in this paper has been implemented for the official analyses of aminoglycosides in eggs as a part of the National Residue Control Plan for the surveillance of veterinary drug residues in food of animal origin as well as in commercial research. So far, no aminoglycosides have been detected in any of the egg samples tested. Additionally, the method described in this study determining 12 aminoglycosides was verified by analyzing the confirmation samples sent in the last year. More than 20 muscle and kidney samples from cattle and pigs were analyzed. Seven kidney samples contained aminoglycosides (NEO, DHSTR) at a concentration of 794-21,950 µg/kg, of which three were non-compliant results above CCα. The demonstrated method will be applied in the future to official control of aminoglycosides in other matrices, as the method developed so far does not cover the need for analyses of new compounds (AMI, APR, HYG, SIS, TOB) and matrices (processed food).
The analytical reference standards of DHSTR, GEN, KAN, NEO, PAR, STR, and SPC were purchased from Dr Ehrenstorfer (Augsburg, Germany), and AMI, APR, HYG, RIB, SIS, TOB from Sigma-Aldrich (St. Louis, MO, USA). Strata X (100 mg, 6 mL), Strata X-CW (100 mg, 6 mL), and Strata X-AW (100 mg, 6 mL) cartridges were from Phenomenex (Torrance, CA, USA). Oasis HLB (60 mg, 3 mL) cartridges were from Waters (Milford, MA, USA). All matrices were obtained from local supermarkets or were originating from the Polish official residue control program. Samples were analyzed to ensure the absence of aminoglycoside residues and kept frozen at −18 • C until use.

Preparation of the Standard Stock Solution and Working Solutions
Each analyte's individual standard stock solutions (1000 µg/mL) were dissolved in a mixture of water/acetonitrile/acetic acid (78:20:2, v/v/v). All standard stock solutions were stored at −18 • C for no longer than 3 months. Working solutions and an internal standard solution (IS) were prepared in ultra-pure water and stored at 4 • C for 1 month.

Method Validation
The presented method was validated according to the Commission Implementing Regulation (EU) 2021/808 of 22 March 2021, repealing Decisions 2002/657/EC and 98/179/EC. The validation process consisted of determining the following parameters: linearity, selectivity, precision, recovery, and decision limit (CCα). The limit of quantification (LOQ) and matrix effect were also assigned. Linearity was determined using a matrix-matched calibration curve prepared by fortifying antibiotic-free matrices at ten concentration levels, depending on the analyte and matrices. Twenty blank samples for different matrices were analyzed for potential disruption with endogenous substances to determine the selectivity. Precision (repeatability and within-laboratory reproducibility) was determined after fortifying six samples on 1, 2, and 3× VL for matrices without MRL, and in the case of designated values of MRL-0.1-0.5, 1, 1.5 MRL, in six replicates at each level. The repeatability was carried out on the same day, instrument, and operator. The coefficient of variations (CV) was calculated. Within-laboratory reproducibility was determined on two days with the same instrument and operators. For trueness, samples were fortified as for precision (1, 2, and 3× VL for matrices without MRL, and in the case of designated values of MRL-0.1-0.5, 1, 1.5 MRL) in six replicates. Trueness (%) was calculated as (mean recovery-corrected concentration detected) × 100/fortification level. The overall CVs were calculated. To test the method's ruggedness, we tested the sample centrifugation temperature, chromatography column temperature, volume of extraction mixture, and concentration of HFBA added to the final extract. The results were analyzed using the Youden test. Decision limits were determined by analyzing 20 blank samples fortified above the MRL (for authorized substances) or VL (for unauthorized substances). The LOQ was established as the lowest point of the matrix calibration curve. The relative matrix effect was calculated for 20 different blank samples at VL. The matrix effect was assessed by calculating the matrix factor (MF) as the ratio of the analytes peak area of the extract fortified after extraction relative to the peak area obtained from the standard solution. The relative matrix effect was calculated as MF (standard) = peak area of MMS standard/peak area of solution standard, where MMS is matrix-matched standard. The calculated CV should not be greater than 20%.

Conclusions
Despite the current trends in the use of newer and less toxic antibiotics, aminoglycosides are still very popular due to their relatively low cost and wide range of action. Awareness is growing about the risks to consumer health of consuming food contaminated with antibiotic residues. Therefore, it is important to control their use with validated and sensitive methods developed for detecting residues in food of animal origin.
As indicated in the literature, this is the very first study on the simultaneous determination of aminoglycosides in as many as thirteen different matrices (muscle, kidney, liver, fat, sausages, shrimps, fish, honey, milk, eggs, whey powder, sour cream, curd). Even though the matrices were different, it was possible to match one technique of aminoglycosides extraction in all tested food materials. Satisfactory validation results confirm that the developed method can be used to analyze aminoglycosides as a part of the National Residue Control Plan for surveillance of veterinary drug residues in food of animal origin.